Elucidation of the Cl-transport mechanism of halorthodopsin by time-resolved electric and spectroscopic measurements

通过时间分辨电学和光谱测量阐明盐紫质的 Cl 传输机制

• 批准号: 11680653
• 负责人: MUNEYUKI Eiro
• 金额: $ 2.37万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680653/
• 关键词: halorhodopsin; ion pump; photocych; photovoltage; spectroscopic measurements; フォトサイクル; 電荷移動

First of all, we improved the photovoltage measurements system so that it enabled us to examine the photovoltage generation from 1 μsec to 500 msec.Then, using the time-resolved photovoltage measurement system, we examined photovoltage kinetics of halorthodopsin from Halobacterium salinarum. As a function of chloride concentrations. The photovoltage consisted of two major components ; one with a sub-ms range time constant and the other with ms range time constant with different amplitudes., These components exhibited different Cl^- concentration dependencies (0.1 M to 9 M). We found that the time constant for the fast component was relatively independent of the Cl^- concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl^- concentrations. The fast and the slow processes were attributed to charge (Cl^-) movements within the protein and related to Cl^- ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that … More they correspond to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl^- concentration dependence and the Cl^- concentration dependence of its time constant suggested a weak and highly cooperative Cl^- binding site(s) at cytoplasmic side (apparent K_D of about 5 M and Hill coefficient ≧ 5). The Cl^- concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be > 100 ms.Electrogenic activity of the halorhodopsin (hR) from shark strain was also examined. After absorbing hR-overproduced membrane fraction onto a thin polymer film, we measured photoelectric current upon illumination at various Cl^- concentrations. The amplitude of the photoelectric current exhibited a bell-shaped Cl^- concentration dependency. It was found that R108Q mutant also exhibited photoelectric current at a high Cl^- concentration. The result indicates that R108 is important for uptaking Cl^- from the medium, but once Cl^- is located near the Schiff base, hR can exhibit electrogenicity even without R108. Less

首先，我们改进了光电压测量系统，使其能够检测从1微秒到500毫秒的光电压产生。然后，使用时间分辨光电压测量系统，我们研究了盐生盐杆菌盐视蛋白的光电压动力学。作为氯化物浓度的函数。光电压由两个主要分量组成;一个具有亚ms范围时间常数，另一个具有不同幅度的ms范围时间常数。这些组分表现出不同的Cl^-浓度依赖性（0.1 M至9 M）。我们发现，快成分的时间常数与Cl^-浓度相对无关，而慢成分的时间常数在Cl^-浓度较高时呈S形增加。快和慢过程分别归因于蛋白质内的电荷（Cl^-）运动和Cl^-排出。SHR-膜悬浮液的激光光解研究表明， ...更多信息 它们对应于N中间体的形成和衰变。慢分量的光电压幅值与Cl^-浓度呈钟形关系，其时间常数与Cl^-浓度的关系表明在胞质侧存在一个弱的、高度合作的Cl^-结合位点（表观K_D约为5 M，Hill系数约为5）。光电压幅度和慢过程时间常数的Cl^-浓度依赖性表明自发弛豫和离子移位之间存在竞争。弛豫时间常数大于100 ms。同时，还研究了鲨鱼卤视紫红质（hR）的生电活性。在将过量产生hR的膜部分吸收到薄聚合物膜上之后，我们测量了在不同Cl^-浓度下照射时的光电流。光电流的振幅呈现出钟形的Cl^-浓度依赖性。发现R108 Q突变体在高Cl^-浓度下也表现出光电流。结果表明，R108对于从培养基中摄取Cl^-是重要的，但是一旦Cl^-位于Schiff碱附近，即使没有R108，hR也可以表现出产电性。少

项目成果

Mitome, N, Ono, S., Suzuki, T., Shimabukuro, K., Muneyuki, E, Yoshida, M.: "Presence of phosphate at a catalytic site suppresses the formation of the MgADP inhibited form of F_1-ATPase"Eur. J. Biochem.. 269. 53-60 (2002)

Mitome, N, Ono, S., Suzuki, T., Shimabukuro, K., Muneyuki, E, Yoshida, M.：“催化位点上磷酸盐的存在会抑制 F_1-ATP 酶的 MgADP 抑制形式的形成”Eur

Yoshida, M. Muneyuki, E., Hisabori, T.: "ATP SYNTHASE-A MARVELLOUS ROTARY ENGINE OF THE CELL"Nature Review Molecular Cell Biology. 2. 669-677 (2001)

Yoshida, M. Muneyuki, E., Hisabori, T.：“ATP 合成酶 - 细胞的奇妙旋转引擎”《自然评论分子细胞生物学》。

Daichi Okuno, Makoto Asaumi, Eiro Muneyuki: "Chloride concentration dependency of the electrogenic activity of halorhodopsin"Biochemistry. 38. 5422-5429 (1999)

Daichi Okuno、Makoto Asaumi、Eiro Muneyuki：“盐视紫红质生电活性的氯化物浓度依赖性”生物化学。

宗行英朗, 吉田賢右: "新ミトコンドリア学 第一章1・3エネルギー転換系と分子モーター"内海耕慥・井上正康 監修 共立出版. 14-21 (2001)

Hideaki Muneyuki、Kensuke Yoshida：“新线粒体科学第 1 1、3 章能量转换系统和分子马达”由 Koki Utsumi 和 Masayasu Inoue Kyoritsu Shuppan 编辑 14-21 (2001)。

Noriyo Mitome, Sakurako Ono, Toshiharu Suzuki, Katsuya Shimabukuro, Eiro Muneyuki, Masasuke Yoshida: "Presence of phosphate at a catalytic site suppresses the formation of the MgADP inhibited form of F_1-ATPase"Eur. J. Biochem.. 269. 53-60 (2002)

Noriyo Mitome、Sakurako Ono、Toshiharu Suzuki、Katsuya Shimabukuro、Eiro Muneyuki、Masuke Yoshida：“催化位点上磷酸盐的存在抑制了 MgADP 抑制形式的 F_1-ATP 酶的形成”Eur。